Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors

Tolerance to self-proteins involves multiple mechanisms, including conventional CD4⁺ T-cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells (nTregs). The significant incidence of autoantibodies specific for the blood coagulation factor VIII (FVIII) in healthy donors illustrates that tolerance to self-proteins is not always complete. In contrast to FVIII-specific Tconvs, FVIII-specific nTregs have never been revealed and characterized. To determine the frequency of FVIII-specific Tregs in human peripheral blood, we assessed the specificity of in vitro expanded Tregs by the membrane expression of the CD137 activation marker. Amplified Tregs maintain high levels of FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg-specific demethylated region. The cells retained FOXP3 expression after long-term culture in vitro, strongly suggesting that FVIII-specific Tregs are derived from the thymus. From eleven healthy donors, we estimated the frequencies of FVIII-specific Tregs at 0.17 cells per million, which is about 10-fold lower than the frequency of FVIII-specific CD4⁺ T cells we previously published. Our results shed light on the mechanisms of FVIII tolerance by a renewed approach that could be extended to other self- or non-self-antigens.     

Phenotypic analysis of human peripheral blood regulatory T cells (CD4+FOXP3+CD127lo/–) ex vivo and after in vitro restimulation with malaria antigens

Regulatory T cells (Treg) play crucial roles in regulating autoimmune responses and immunity to tumors and infectious diseases. However, numerous subpopulations of Treg are now being described and the utility of various Treg markers is being reassessed. Here we report the results of a detailed phenotypic comparison of two supposedly regulatory human T‐cell populations, namely CD4⁺FOXP3⁺ T cells and CD4⁺CD25^hi T cells. We find that CD4⁺FOXP3⁺ cells are extremely heterogeneous with respect to CD25 expression and that FOXP3⁺ and CD25^hi CD4⁺ T cells differ in their expression of chemokine receptors (CCR), CD95 and Bcl‐2, suggestive of distinct migration characteristics and susceptibility to apoptosis. Further, we propose that CD25 expression should be regarded as an activation marker rather than as a defining marker of Treg. Lastly, CD4⁺FOXP3⁺ T cells activated in vitro with malaria antigen expressed the highest levels of CCR4 and CD95, and the lowest levels of CCR7, indicating that they are most likely generated from effector memory cells during an immune response and rapidly succumb to apoptosis at the end of the response.     

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

The adoptive transfer of CD4⁺CD25⁺ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127⁺ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3⁺ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4⁺CD25⁺CD127^low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA^?FOXP3⁺ memory‐type Treg as the main source of converting cells, whereas CD45RA⁺FOXP3⁺ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.     

Surface expression of CD39 identifies an enriched Treg‐cell subset in the rheumatic joint,which does not suppress IL‐17A secretion

Treg cells are important for the maintenance of self‐tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP‐hydrolyzing enzymes CD39 and CD73 and the resulting anti‐inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3⁺ Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3⁺ Treg cells displayed high expression levels of rate‐limiting CD39, whereas CD73 was diminished. FOXP3⁺CD39⁺ Treg cells were also abundant in synovial tissue. Furthermore, FOXP3⁺CD39⁺ Treg cells did not secrete the proinflammatory cytokines IFN‐γ and TNF after in vitro stimulation in contrast to FOXP3⁺CD39^? T cells. FOXP3⁺CD39⁺ Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg‐specific demethylated region and coculture assays confirmed that CD25⁺CD39⁺ T cells have suppressive capacity, while their CD39^? counterparts do not. Overall, our data show that FOXP3⁺CD39⁺ Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T‐cell functions including production of IFN‐γ, TNF, and IL‐17F but do not limit IL‐17A secretion.     

Functional islet‐specific Treg can be generated from CD4+CD25− T cells of healthy and type 1 diabetic subjects

CD4⁺CD25⁺FOXP3⁺ Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self‐antigen‐specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen‐specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen‐specific adaptive Treg were induced in vitro by activation of CD4⁺FOXP3^? T cells with glutamic acid decarboxylase and islet‐specific glucose‐6‐phosphate catalytic subunit‐related protein peptides in the context of T1D‐associated HLA‐DRβ alleles. Antigen‐specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza‐specific Treg from the same study population. The function of tetramer⁺ cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet‐specific Treg may potentially be used as a targeted immunotherapy in T1D.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.     

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus

Thymic commitment of human FOXP 3⁺ regulatory T cells begins at the double‐positive (DP ) CD 4⁺ CD 8⁺ stage. In the current study, we show that interleukin‐2 promotes the development of FOXP 3⁺ thymocytes and enhances their survival at the DP phase. IL ‐2 increases the frequency of FOXP 3⁺ cells and promotes the Treg phenotype after TCR ‐mediated positive selection at the most mature DP stage. However, it has no effect on FOXP 3⁺ cells at the earlier maturation steps before positive selection. DP FOXP 3⁺ thymocytes are highly susceptible to cell death but IL ‐2 promotes their survival. The anti‐apoptotic protein BCL ‐2 (B Cell Lymphoma 2) is also upregulated by IL ‐2 at the most mature DP stage. In addition to IL ‐2, we identify IL ‐15 to have a significant role in the upregulating FOXP 3 and survival of Tregs at the DP phase. IL ‐7 also increases the expression of BCL ‐2 in the DP FOXP 3⁺ thymocytes. Our results indicate that common gamma chain cytokines IL ‐2, IL ‐7 and IL ‐15 promote the development of regulatory T cells at the most mature DP stage after TCR ‐mediated positive selection through suppressing cell death.     

CD39 is involved in mediating suppression by Mycobacterium bovis BCG‐activated human CD8+CD39+ regulatory T cells

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4⁺ Treg cells, relatively little is known about the phenotype and function of CD8⁺ Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E‐NTPDase1) on Mycobacterium‐activated human CD8⁺ T cells. These CD8⁺CD39⁺ T cells significantly co‐expressed the Treg markers CD25, Foxp3, lymphocyte activation gene‐3 (LAG‐3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen‐specific CD4⁺ T helper‐1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8⁺ T cells and indicate that CD39 is functionally involved in suppression by CD8⁺ Treg cells.     

Deacetylase inhibition increases regulatory T cell function and decreases incidence and severity of collagen-induced arthritis

Collagen-induced arthritis (CIA) is an established mouse model of disease with hallmarks of clinical rheumatoid arthritis. Histone/protein deacetylase inhibitors (HDACi) are known to inhibit the pathogenesis of CIA and other models of autoimmune disease, although the mechanisms responsible are unclear. Regulatory T cell (Treg) function is defective in rheumatoid arthritis. FOXP3 proteins in Tregs are present in a dynamic protein complex containing histone acetyltransferase and HDAC enzymes, and FOXP3 itself is acetylated on lysine residues. We therefore investigated the effects of HDACi therapy on regulatory T cell function in the CIA model. Administration of an HDACi, valproic acid (VPA), significantly decreased disease incidence (p < 0.005) and severity (p < 0.03) in CIA. In addition, VPA treatment increased both the suppressive function of CD4⁺CD25⁺ Tregs (p < 0.04) and the numbers of CD25⁺FOXP3⁺ Tregs in vivo. Hence, clinically approved HDACi such as VPA may limit autoimmune disease in vivo through effects on the production and function of FOXP3⁺ Treg cells.     

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg

CD25⁺ FOXP3⁺CD4⁺ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high‐level expression of FOXP3 (FOXP3^high) is sufficient to confer suppressive activity to normal non‐Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3^high but not FOXP3^low Treg. Such VEGFR2⁺ Treg exist in several tissues including PBMC and malignant effusion‐derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.     

Dexamethasone upregulates FOXP3 expression without increasing regulatory activity

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are “truly” Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4⁺CD25^high cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25^high cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4⁺CD25^high cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.     

Freeze-thaw lysates of Plasmodium falciparum-infected red blood cells induce differentiation of functionally competent regulatory T cells from memory T cells

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4⁺ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen‐derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time‐ and dose‐dependent manner without the addition of exogenous costimulatory factors. The PfSE‐mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE‐labeled Treg cells identified CD4⁺CD45RO⁺CD25^? memory T cells rather than Treg cells as the primary source of PfSE‐induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE‐induced effector T cells.